Aberrant activation of NOTCH signaling due to the presence of activating mutations in the NOTCH1 receptor play a critical role in the pathogenesis in more than 50% human T-cell lymphoblastic leukemias (T-ALL). The identification of NOTCH1 mutations of T-ALL cases has prompted the initiation of clinical trials to test the effectiveness of blocking NOTCH1 signaling with ?-secretase inhibitors (GSIs) in this disease. However, GSIs are active only in a small fraction of T-ALLs with NOTCH1 mutations, suggesting that mechanisms responsible for GSI resistance may limit the effectiveness of GSIs in T-ALL. Our preliminary data shows that: (i) NOTCH1 upregulates the PI3K/AKT pathway in T-ALL;(ii) NOTCH1 signaling downregulates the expression of the PTEN tumor suppressor gene;and (iii) loss of PTEN is associated with resistance to NOTCH1 inhibition with GSIs. Thus, our central hypothesis is that the activity of the PI3K-AKT pathway mediates, at least in part, the oncogenic effects of aberrant NOTCH1 signaling. We further propose that constitutive activation of PI3K-AKT due to mutational loss of PTEN promotes cell growth proliferation and survival rendering T-ALL cells insensitive to NOTCH1 inhibition. The short-term goals of this research are to establish the significance of the interaction between NOTCH1 and PI3K/AKT signaling pathways in T-cell transformation and therapy response. To achieve these objectives, we propose the following specific aims: Aim 1. To analyze the interaction between NOTCH1 and Pten-PI3K-AKT in T-cell transformation. In this aim we will use retroviral oncogene transfer to express activated NOTCH1 in hematopoietic progenitors from wild type and Pten knockout mice to assess the relevance of the interaction between oncogenic NOTCH1 and PI3K-Akt signaling in the pathogenesis of T-cell lymphoblastic tumors. Aim 2. To analyze the functional relationship between NOTCH1 and Pten in leukemia stem cells. In this aim we will analyze the role of the NOTCH1 and PI3K-Akt signaling pathways in the homeostasis of the leukemia stem cells from Pten-wild type, Pten-haploinsuficient and Pten-null tumors expressing activated NOTCH1. Aim 3. To analyze the interaction between NOTCH and Akt signaling in the response of T-ALL to molecularly targeted drugs. Does Pten-loss induce resistance to NOTCH1 inhibitors? Does inhibition of the AKT signaling pathway reverse resistance to NOTCH1 inhibitors? Do Pten-heterozygous and/or Pten-null T-ALL tumors show increased sensitivity to Akt inhibitors or to drugs targeting downstream effectors of Akt signaling such as mTOR, NFKB and FOXO factors? PUBLIC HEALTH RELEVANCE: This project aims to analyze the genetic interaction between NOTCH1 and PI3K-AKT pathways in the pathogenesis of T-cell lymphoblastic leukemias (T-ALL) and their role in tumor resistance to emerging therapies targeting the NOTCH1 and AKT oncoproteins. The experiments outlined here will set the basis for the rational development of new therapies against T-ALL.